Showkat Ahmad Ganie

Identification and characterization of salt responsive miRNA-SSR markers in rice (Oryza sativa)

Salinity is an important abiotic stress that affects agricultural production and productivity. It is a complex trait that is regulated by different molecular mechanisms. miRNAs are non-coding RNAs which are highly conserved and regulate gene expression. Simple sequence repeats (SSRs) are robust molecular markers for studying genetic diversity. Although several SSR markers are available now, challenge remains to identify the trait-specific SSRs which can be used for marker assisted breeding. In order to understand the genetic diversity of salt responsive-miRNA genes in rice, SSR markers were mined from 130 members of salt-responsive miRNA genes of rice and validated among the contrasting panels of tolerant as well as susceptible rice genotypes, each with 12 genotypes. Although 12 miR-SSRs were found to be polymorphic, only miR172b-SSR was able to differentiate the tolerant and susceptible genotypes in 2 different groups. It had also been found that miRNA genes were more diverse in susceptible genotypes than the tolerant one (as indicated by polymorphic index content) which might interfere to form the stem-loop structure of premature miRNA and their subsequent synthesis in susceptible genotypes. Thus, we concluded that length variations of the repeats in salt responsive miRNA genes may be responsible for a possible sensitivity to salinity adaptation. This is the first report of characterization of trait specific miRNA derived SSRs in plants.

Identification and analysis of novel salt responsive candidate gene based SSRs (cgSSRs) from rice (Oryza sativa L.).

BackgroundMajority of the Asian people depend on rice for nutritional energy. Rice cultivation and yield are severely affected by soil salinity stress worldwide. Marker assisted breeding is a rapid and efficient way to develop improved variety for salinity stress tolerance. Genomic microsatellite markers are an elite group of markers, but there is possible uncertainty of linkage with the important genes. In contrast, there are better possibilities of linkage detection with important genes if SSRs are developed from candidate genes. To the best of our knowledge, there is no such report on SSR markers development from candidate gene sequences in rice. So the present study was aimed to identify and analyse SSRs from salt responsive candidate genes of rice.ResultsIn the present study, based on the comprehensive literature survey, we selected 220 different salt responsive genes of rice. Out of them, 106 genes were found to contain 180 microsatellite loci with, tri-nucleotide motifs (56%) being most abundant, followed by di-(41%) and tetra nucleotide (2.8%) motifs. Maximum loci were found in the coding sequences (37.2%), followed by in 5′UTR (26%), intron (21.6%) and 3′UTR (15%). For validation, 19 primer sets were evaluated to detect polymorphism in diversity analysis among the two panels consisting of 17 salt tolerant and 17 susceptible rice genotypes. Except one, all primer sets exhibited polymorphic nature with an average of 21.8 alleles/primer and with a mean PIC value of 0.28. Calculated genetic similarity among genotypes was ranged from 19%-89%. The generated dendrogram showed 3 clusters of which one contained entire 17 susceptible genotypes and another two clusters contained all tolerant genotypes.ConclusionThe present study represents the potential of salt responsive candidate gene based SSR (cgSSR) markers to be utilized as novel and remarkable candidate for diversity analysis among rice genotypes differing in salinity response.

Promoter methylation regulates the abundance of osa-miR393a in contrasting rice genotypes under salinity stress

MicroRNAs (miRNAs) are important molecules that regulate gene expression under salinity stress. Despite their evolutionary conservation, these regulatory elements have been shown to behave differently in different plant species under a particular environmental stress. In this study, we investigated the behavior of salt responsive osa-miR393a and its target gene (TIR1, LOC_Os05g05800) in salt-tolerant (FL478) and salt-sensitive (IR29) rice genotypes. It was found that the mature and precursor sequences of osa-miR393a as well as its cleavage site in TIR1 were conserved among salt tolerant and sensitive genotypes. Promoters of different salt-responsive miRNAs were also found to be less variable between salt-tolerant and salt-susceptible genotypes. Analysis of gene expression, promoter methylation, and cis-element abundance showed that osa-miR393a behaves differently in FL478 and IR29. Salt stress altered the expression pattern of osa-miR393a-TIR1 module in a time-dependent manner in the roots and shoots of two genotypes. Promoter methylation of this regulatory module was also altered at different time points under salt stress. Expression analysis in two genotypes indicated the overall down-regulation of osa-miR393a and up-regulation of TIR1 in FL478 and their reciprocal regulation in IR29. The expression results were complemented by the differential promoter methylation and cis-element abundance of this regulatory module. Together, the results of transcript abundance and promoter methylation of osa-miR393a-TIR1 module signified the association between these two processes which is reported for the first time in plants to the best of our knowledge.

Genome-wide development of novel miRNA-based microsatellite markers of rice (Oryza sativa) for genotyping applications

AbstractRice (Oryza sativa L.) plays a crucial role in assuring food security to the world’s population and is recognized as a superior model plant of monocot crops. Molecular markers, especially co-dominant markers such as simple sequence repeats (SSRs), play an important role in marker-assisted breeding of rice. Although a large number of SSRs are available, most of them are either from protein-coding regions or untranslated regions of the rice genome. SSRs from non-coding regions of the rice genome are almost undiscovered, but discovery of new SSR markers remains a challenge for molecular breeders. The development of novel markers from the conserved regions of different genomes will thus be useful for studying the genetic diversity of closely related species or self-pollinated species. In the present study, a genome-wide investigation yielded a total of 129 SSR markers from the conserved microRNA (miRNA) genes of rice, and 20 of them with repeat numbers ≥7, from all the 12 chromosomes, were validated among 24 diverse rice genotypes. The highest number of repeat motifs corresponded to di-nucleotides, while the tetra-nucleotides were repeated the lowest number of times. Our results therefore show that the number of repeats is inversely related to the length of repeat. The genetic diversity was found to be higher among the exotic material than the Indian landraces, as indicated by their polymorphic information content values of 0.31 and 0.29, respectively. The results hence showed that the miRNA-based microsatellite marker system is a very proficient, novel and breeder-friendly source for genetic diversity analysis or genotyping of rice. The present study is the first report on genome-wide identification and characterization of rice miRNA-SSRs.

Identification of Novel and Conserved miRNAs from Extreme Halophyte, Oryza coarctata, a Wild Relative of Rice

Oryza coarctata, a halophyte and wild relative of rice, is grown normally in saline water. MicroRNAs (miRNAs) are non-coding RNAs that play pivotal roles in every domain of life including stress response. There are very few reports on the discovery of salt-responsive miRNAs from halophytes. In this study, two small RNA libraries, one each from the control and salt-treated (450 mM NaCl for 24 h) leaves of O. coarctata were sequenced, which yielded 338 known and 95 novel miRNAs. Additionally, we used publicly available transcriptomics data of O. coarctata which led to the discovery of additional 48 conserved miRNAs along with their pre-miRNA sequences through in silico analysis. In total, 36 known and 7 novel miRNAs were up-regulated whereas, 12 known and 7 novel miRNAs were down-regulated under salinity stress. Further, 233 and 154 target genes were predicted for 48 known and 14 novel differentially regulated miRNAs respectively. These targets with the help of gene ontology analysis were found to be involved in several important biological processes that could be involved in salinity tolerance. Relative expression trends of majority of the miRNAs as detected by real time-PCR as well as predicted by Illumina sequencing were found to be coherent. Additionally, expression of most of the target genes was negatively correlated with their corresponding miRNAs. Thus, the present study provides an account of miRNA-target networking that is involved in salinity adaption of O. coarctata.

Assessment of genetic diversity of Saltol QTL among the rice (Oryza sativa L.) genotypes

Eight Saltol quantitative trait locus (QTL) linked simple sequence repeat (SSR) markers of rice (Oryza sativa L.) were used to study the polymorphism of this QTL in 142 diverse rice genotypes that comprised salt tolerant as well as sensitive genotypes. The SSR profiles of the eight markers generated 99 alleles including 20rare alleles and 16 null alleles. RM8094 showed the highest number (13) of alleles followed by RM3412 (12), RM562 (11), RM493 (9) and RM1287 (8) while as, RM10764 and RM10745 showed the lowest number (6) of alleles. Based on the highest number of alleles and PIC value (0.991), we identified RM8094 as suitable marker for discerning salt tolerant genotypes from the sensitive ones. Based upon the haplotype analysis using FL478 as a reference (salt tolerant genotypes containing Saltol QTL), we short listed 68 rice genotypes that may have at least one allele of FL478 haplotype. Further study may confirm that some of these genotypes might have Saltol QTL and can be used as alternative donors in salt tolerant rice breeding programmes.

Cloning and in silico analysis of a gene encoding a putative β-carbonic anhydrase from cowpea (Vigna unguiculata L. Walp)

A gene encoding putative β-carbonic anhydrase gene (CA) from cowpea (Vigna unguiculata L. Walp.) was characterized and designated as VuCA1 (GenBank ID: JQ429799). While the genomic sequence of CA was found to be 1470 bp long with 4 introns, the open reading frame was 990 bp in length. The deduced amino acid sequence of CA contained two characteristic conserved domains, i.e. CSDSRV and EYAVLHLKVSNIVVIGHSACG, which showed high degree of homology with other β-CA genes of angiosperms. We have reported three-dimensional model of VuCA1 and quality of the predicted model was analysed with PROCHECK. Molecular docking of modeled VuCA1 revealed similar binding pockets for different substrates and products. The expression of VuCA1 transcripts was detected in different parts of the cowpea plant, with highest level observed in leaves, followed by flower buds, weak expression in stems, and the weakest in roots. The expression of VuCA1 transcripts of leaves was downregulated by zinc deficiency but upregulated by salt and dehydration stress.

Comprehensive survey and evolutionary analysis of genome-wide miRNA genes from ten diploid Oryza species

BackgroundMicroRNAs (miRNAs) are non-coding RNAs that play versatile roles in post-transcriptional gene regulation. Although much is known about their biogenesis, and gene regulation very little is known about their evolutionary relation among the closely related species.ResultAll the orthologous miRNA genes of Oryza sativa (japonica) from 10 different Oryza species were identified, and the evolutionary changes among these genes were analysed. Significant differences in the expansion of miRNA gene families were observed across the Oryza species. Analysis of the nucleotide substitution rates indicated that the mature sequences show the least substitution rates among the different regions of miRNA genes, and also show a very much less substitution rates as compared to that of all protein-coding genes across the Oryza species. Evolution of miRNA genes was also found to be contributed by transposons. A non-neutral selection was observed at 80 different miRNA loci across Oryza species which were estimated to have lost ~87% of the sequence diversity during the domestication. The phylogenetic analysis revealed that O. longistaminata diverged first among the AA-genomes, whereas O. brachyantha and O. punctata appeared as the eminent out-groups. The miR1861 family organised into nine distinct compact clusters in the studied Oryza species except O. brachyantha. Further, the expression analysis showed that 11 salt-responsive miRNAs were differentially regulated between O. coarctata and O. glaberrima.ConclusionOur study provides the evolutionary dynamics in the miRNA genes of 10 different Oryza species which will support more investigations about the structural and functional organization of miRNA genes of Oryza species.

Erratum to: Genome-wide Analysis of Zinc Transporter Genes of Maize (Zea mays)

Zinc (Zn) is an essential micronutrient for plants and animals. Zinc-regulated transporters and iron-regulated transporter-like proteins (ZIP) are important zinc transporters in plants with the characteristic ZIP domain (Pfam:PF02535). Although individual genes belonging to the ZIP family had been discovered in various plants, genome-wide analysis of the paralouges (ZmZIP) in maize and their relationships with other related genera has so far not been conducted. We performed a genome-wide analysis and identified 12 members of the ZIP gene family in maize. Chromosomal locations, motif organization, and biochemical characterizations of proteins, as well as exon–intron, trans-membrane domains of these ZmZIP genes were determined, which indicated the structural diversity of ZmZIP. Additionally, apart from the identification of the canonical form of the metal binding signature in ZIP domains of the ZmZIP proteins, we also identified a new conserved plant ZIP signature. Further, tissue-specific expressions of those genes were determined by real-time PCR in the flag leaf as well as in 10-day-old-baby kernel among the high and low kernel zinc-containing maize inbreds. We found that overall transcript abundance was higher in the flag leaf than the kernel in both the inbreds for all the members except two, namely ZmZIP5 and ZmZIP11 were expressed more in flag leaf of a high-kernel zinc-containing inbreds than a lowkernel zinc-containing inbreds. Therefore, these results provide a basis for further functional characterization of specific ZmZIP genes in the future.